Project Summary/Abstract Numerous pathogenic viruses utilize a lympho-hematogenous route of dissemination upon penetration of their natural host epithelial surfaces. These viruses replicate in the lymph nodes (LN) before spreading to distant organs, resulting in the induction of systemic disease. Ectromelia virus (ECTV) is widely utilized as the prototypical virus for studying lympho-hematogenous spreading. Understanding the mechanisms of viral dissemination and host immune-virus interactions is paramount to not only our understanding of how humans control the dissemination of their own natural pathogens but is critical for the advancement of vaccine development and efficacy. Work from our lab have supported the concept that LNs are not only sites that facilitate lymphocyte priming but also serve as principal sites where innate and adaptive cells curtail the systemic propagation of pathogens. We have recently demonstrated that inflammatory monocytes (iMo) rapidly migrate to the draining LN (dLN) after infection and play a critical role in curtailing viral replication and dissemination. They are the principal producers of type I interferon (T1-IFN) in the dLN and their accumulation to the dLN is dependent on extrinsic TLR9/MyD88 expression, specifically by CCL7/CCL2 producing CD11c+ cells. Our preliminary data indicates that these CD11c+ cells are skin-derived, migratory dendritic cells (skin- mDCs) from the skin of the footpad (the initial site of virus breach and infection). TLR9/MyD88 deficiency as well as localized pertussis toxin treatment ablates skin-mDC migration to the dLN, resulting in a concomitant decrease in iMo accumulation and higher viral replication, indicating an important role for viral sensing mechanisms intrinsic to these skin-mDCs. However, the mechanisms of viral transport from the initial site of infection to the dLN is not well defined and furthermore, the underlying mechanisms through which TLR9 and MyD88 control skin-mDC function is unknown. Therefore, the proposed research will aim to 1.) elucidate the skin-mDC-dependent and ?independent mechanisms governing viral transport as well as define the specific effector functions of the skin-mDC subsets during viral infection 2.) delineate the TLR9 and MyD88-intrinsic mechanisms that dictate skin-mDC migration and function. Overall, our experiments will contribute to the better understanding of the early anti-viral response in the skin to a pathogen that spreads lymphohematogenously in its natural host. This is especially critical given that the degree of effectiveness of the initial host response plays an important role in dictating the efficacy of the entire host response. This will help contribute to a better understanding of how humans control the dissemination of their own natural pathogens.